title: Increased contractility of hepatic stellate cells in cirrhosis is mediated by enhanced Ca2+-dependent and Ca2+-sensitization pathways.
abstract: Activation of hepatic stellate cells (HSCs) results in cirrhosis and portal hypertension due to intrahepatic resistance. Activated HSCs increase their contraction after receptor agonist stimulation; however, the signaling pathways for the regulation of contraction are not fully understood. The aim of this study was to elucidate the change in contractile mechanisms of HSCs after cirrhotic activation. The expression pattern of contractile regulatory proteins was analyzed with quantitative RT-PCR and Western blotting. The phosphorylation levels of myosin light chain (MLC), 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), and MLC phosphatase targeting subunit 1 (MYPT1) after endothelin-1 (ET-1) stimulation in culture-activated HSCs were measured using phosphorylation-specific antibodies. In vivo-activated HSCs were isolated from rats subjected to bile duct ligation and repeated dimethylnitrosoamine injections. HSCs showed increased expression of not only α-smooth muscle actin, but also the contractile regulatory proteins MLC kinase (MLCK), Rho kinase 2 (ROCK2), and CPI-17 during HSC activation in vitro. In culture-activated HSCs, ET-1 increased phosphorylation of CPI-17 at Thr18, which was markedly inhibited by the PKC inhibitor Ro-31-8425. ET-1 induced phosphorylation of MYPT1 at Thr853, which was suppressed by the ROCK inhibitor Y-27632. ET-1 induced sustained phosphorylation of MLC at Thr18/Ser19, which was inhibited by both Ro-31-8425 and Y-27632. Consistent with the data obtained from the in vitro study, HSCs isolated from cirrhotic rats showed increased expression of α-smooth muscle actin, MLCK, CPI-17, and ROCK2 compared with HSCs from nontreated rats. Furthermore, MLC phosphorylation in in vivo-activated HSCs was increased, according to enhanced phosphorylation of CPI-17 and MYPT1 in the presence of ET-1. These results suggest that activated HSCs may participate in constriction of hepatic sinusoids in the cirrhotic liver through both Ca(2+)-dependent (MLCK pathway) and Ca(2+)-sensitization mechanism (CPI-17 and MYPT1 pathways).
authors.affiliation: Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Japan.
authors.forename: Masateru
authors.initials: M
authors.lastname: Iizuka
authors.forename: Takahisa
authors.initials: T
authors.lastname: Murata
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Hori
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ozaki
mesh_terms: D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000378:metabolism; D055166:Hepatic Stellate Cells / Q000187:drug effects / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008106:Liver Cirrhosis, Experimental / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D009124:Muscle Proteins / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054645:Protein Phosphatase 1 / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D000199:Actins; D000577:Amides; D002118:Calcium; D019332:Endothelin-1; D007211:Indoles; D008301:Maleimides; D009124:Muscle Proteins; D018994:Myosin Light Chains; D009219:Myosin-Light-Chain Kinase; D010750:Phosphoproteins; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D054645:Protein Phosphatase 1; D011725:Pyridines; D054460:rho-Associated Kinases; C491519:Ppp1r14a protein, rat; C498862:Acta2 protein, rat; C071924:Ro 31-8425; C108830:Y 27632; C499850:Ppp1r12a protein, rat